Secretion of the fungal toxin candidalysin is dependent on conserved precursor peptide sequences.

The opportunistic fungal pathogen Candida albicans damages host cells via its peptide toxin, candidalysin. Before secretion, candidalysin is embedded in a precursor protein, Ece1, which consists of a signal peptide, the precursor of candidalysin and seven non-candidalysin Ece1 peptides (NCEPs), and is found to be conserved in clinical isolates. Here we show that the Ece1 polyprotein does not resemble the usual precursor structure of peptide toxins. C. albicans cells are not susceptible to their own toxin, and single NCEPs adjacent to candidalysin are sufficient to prevent host cell toxicity. Using a series of Ece1 mutants, mass spectrometry and anti-candidalysin nanobodies, we show that NCEPs play a role in intracellular Ece1 folding and candidalysin secretion. Removal of single NCEPs or modifications of peptide sequences cause an unfolded protein response (UPR), which in turn inhibits hypha formation and pathogenicity in vitro. Our data indicate that the Ece1 precursor is not required to block premature pore-forming toxicity, but rather to prevent intracellular auto-aggregation of candidalysin sequences.

Candidalysin Is the Hemolytic Factor of Candida albicans.

Candida albicans produces an important virulence factor, the hypha-associated Ece1-derived secreted peptide toxin candidalysin, which is crucial for the establishment of mucosal and systemic infections. C. albicans has also long been known to be hemolytic, yet the hemolytic factor has not been clearly identified. Here, we show that candidalysin is the hemolytic factor of C. albicans. Its hemolytic activity is modulated by fragments of another Ece1 peptide, P7. Hemolysis by candidalysin can be neutralized by the purinergic receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). PPADS also affects candidalysin's ability to intercalate into synthetic membranes. We also describe the neutralization potential of two anti-candidalysin nanobodies, which are promising candidates for future anti-Candida therapy. This work provides evidence that the historically proposed hemolytic factor of C. albicans is in fact candidalysin and sheds more light on the complex roles of this toxin in C. albicans biology and pathogenicity.

Selenium biofortification of different varieties of apples (Malus domestica) - Influence on protein content and the allergenic proteins Mal d 1 and Mal d 3.

As allergy towards apples is widespread, the evaluation of various cultivation and postharvest influences on the allergenic potential is of great importance. Therefore, the analysis of the Mal d 1 content was the focus of this study, originally dealing with investigating the influence of a selenium biofortification on apple quality. The Mal d 1 content of apples was in most cases reduced when the fruits were biofortified with selenium. Apple variety and climatic conditions were identified as further influencing factors for the Mal d 1 content of the fruits. The separate analysis of the peel and the fruit flesh showed that the content of Mal d 1 in the fruit flesh was significantly lower in the biofortified samples than in the controls. In conclusion, the results indicate that the selenium biofortification of apples and biochemical mechanism behind can reduce the allergenic potential regarding the content of Mal d 1.

Relationship between Phenolic Compounds, Antioxidant Properties, and the Allergenic Protein Mal d 1 in Different Selenium-Biofortified Apple Cultivars (Malus domestica).

Notable parts of the population in Europe suffer from allergies towards apples. To address this health problem, the analysis of the interactions of relevant allergens with other substances such as phenolic compounds is of particular importance. The aim of this study was to evaluate the correlations between the total phenolic content (TPC), polyphenol oxidase (PPO) activity, antioxidant activity (AOA), and the phenolic compound profile and the content of the allergenic protein Mal d 1 in six apple cultivars. It was found that the PPO activity and the content of individual phenolic compounds had an influence on the Mal d 1 content. With regard to the important constituents, flavan-3-ols and phenolic acids, it was found that apples with a higher content of chlorogenic acid and a low content of procyanidin trimers and/or epicatechin had a lower allergenic potential. This is probably based on the reaction of phenolic compounds (when oxidized by the endogenous PPO) with proteins, thus being able to change the conformation of the (allergenic) proteins, which further corresponds to a loss of antibody recognition. When apples were additionally biofortified with selenium, the composition of the apples, with regard to TPC, phenolic profile, AOA, and PPO, was significantly affected. Consequently, this innovative agronomic practice seems to be promising for reducing the allergenic potential of apples.

The Beauty of Asymmetric Membranes: Reconstitution of the Outer Membrane of Gram-Negative Bacteria.

The architecture of the lipid matrix of the outer membrane of Gram-negative bacteria is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is built up by glycolipids. For most Gram-negative species, these glycolipids are lipopolysaccharides (LPS), for a few species, however, glycosphingolipids. We demonstrate experimental approaches for the reconstitution of these asymmetric membranes as (i) solid supported membranes prepared by the Langmuir-Blodgett technique, (ii) planar lipid bilayers prepared by the Montal-Mueller technique, and (iii) giant unilamellar vesicles (GUVs) prepared by the phase transfer method. The asymmetric GUVs (aGUVs) composed of LPS on one leaflet are shown for the first time. They are characterized with respect to their phase behavior, flip-flop of lipids and their usability to investigate the interaction with membrane active peptides or proteins. For the antimicrobial peptide LL-32 and for the bacterial porin OmpF the specificity of the interaction with asymmetric membranes is shown. The three reconstitution systems are compared with respect to their usability to investigate domain formation and interactions with peptides and proteins.

Influence of a Selenium Biofortification on Antioxidant Properties and Phenolic Compounds of Apples (Malus domestica).

Biofortified apples seem to be a suitable produce. In this study, different selenium forms and application levels were applied to the two apple varieties 'Golden Delicious' and 'Jonagold', grown in the years 2017 and 2018 in order to increase the selenium uptake within a typical Western diet. It was shown that the biofortification, which was performed as a foliar application implemented in usual calcium fertilization, led to significantly increased selenium contents in the fruits. Furthermore, biofortification affected the total phenolic content (TPC), the polyphenol oxidase activity (PPO), as well as the antioxidant activity (AOA), the latter measured with the two well-known assays Trolox Equivalent Antioxidant Capacity Assay (TEAC) and Oxygen Radical Absorbance Capacity Assays (ORAC). The varying selenium forms and application levels showed a differing influence on the parameters mentioned before. Higher fertilizer levels resulted in higher selenium accumulation. It was found that PPO activity fluctuates less in biofortified apples. With regard to TPC, selenate led to higher amounts when compared to the untreated controls and selenite resulted in lower TPC. AOA analysis showed no clear tendencies as a result of the selenium biofortification. In the case of 'Jonagold', a higher AOA was generally measured when being biofortified, whereas, in the case of 'Golden Delicious', only one form of application led to higher AOA. Additionally, differences in the amount of major phenolic compounds, measured with High Performance Liquid Chromatography Mass Spectrometry (HPLC-DAD-ESI-MSn), were observed, depending on the conditions of the biofortification and the variety.

Antioxidant Activity and Phenolic Profile of Selected Organic and Conventional Honeys from Poland.

Honey is a natural food product hypothesized to have significant health-beneficial value. The results of recent studies indicate that the biological activity of honey can also be ascribed to phenolic compounds and their antioxidant activity. The aims of this study were: To determine the phenolic profiles of several varieties of Polish honey and their correlation with various factors influencing the quality of honey, plus to verify the impact of production method (organic/conventional) and the pollen content on these profiles. In total, 11 organic and 11 conventional honey samples from Poland were investigated. The botanical origin of the samples was identified through melissopalynological analysis, whereas individual phenolic compounds were determined by the LC/MS analysis. The Folin-Ciocalteau assay was used for the determination of the total phenolic content (TPC). Moreover, the CIE L*a*b* color values were measured and matched with the above-mentioned parameters. The results of the study contribute to the discussion on the health benefits of organic farming. It was found that chrysin may act as a potential indicator compound. The study confirms the existence of the link between TPC and color, and it shows that there is a correlation between pinocembrin and galangin, two compounds that are reported to ameliorate insulin resistance.